Project Summary The primary research goal of this Pathway to Independence proposal is to understand and characterize the mechanisms underlying intrinsic expression of interferon stimulated genes (ISGs) in stem cells. The maintenance of healthy stem cells is essential for tissue repair within an organism. Unlike terminally differentiated cells, however, stem cells do not produce the same robust interferon (IFN) response to combat infection. The mechanisms by which stem cells potently block viral infection are still poorly understood. Our recent discoveries demonstrate that stem cells have high basal levels of cell type-specific subsets of ISGs that confer potent protection against a number of viruses. The mechanisms underlying this intrinsic ISG expression remain elusive. To this end, in the mentored phase of this K99 award, the candidate will be trained in epigenome techniques and bioinformatics to globally define chromatin accessibility associated with intrinsically expressed ISGs in stem cells. As transcription factors (TFs) binding is often associated with these accessible chromatin domains, the analysis of such coordination would allow him to identify TFs driving ISG expression in stem cells. In addition, the candidate will receive training on CRISPR gene knockout screens, and further define candidate TFs with these loss-of-function complementary experiments. Finally, the candidate will be guided on detailed mechanistic characterization of newly identified TFs as well as IRF1, a TF specifically regulates intrinsic ISG expression in hematopoietic stem cells. Additional components of the candidate's comprehensive career development plan are courses and seminars in ethics and grant writing. The training phase will be carried out in the laboratory of Dr. Charles Rice at The Rockefeller University (RU), one of the world's leading laboratories in virology and immunology research. In addition to the significant resources and basic science expertise in this laboratory, the candidate will also benefit from RU's vibrant research community. A critical component of career development will be the close counsel of a highly experienced Advisory Committee, composed of Dr. Charles Rice, Dr. C. David Allis, and Dr. Robert Roeder (all RU), and Dr. David Levy (New York University). The innovative skills and comprehensive datasets obtained in the K99 training phase will set the stage for detailed understanding of the transcriptional networks that regulate intrinsic ISG expression, and more importantly, antiviral resistance in stem cells. This will be key to succeed as a young independent investigator in this highly competitive field of research. In all, the training will fulfill both the candidate's short-term goals of adding new technologies, skills, and experience to his portfolio, and his long- term goals, to become an independent investigator with a research focus on understanding important processes at the heart of host-virus interactions. This study will have important implications for our understanding of stem cell biology, primordial aspects in development and cancer, and the evolution of vertebrate pathogen defense.